Preparation of N2-Ethyl-2′-deoxyguanosine-d4 as an Internal Standard for the Electrospray Ionization-Tandem Mass Spectrometric Determination of DNA Damage by Acetaldehyde

The deuteration of N-2-ethyl-2'-deoxyguanosine (Et-dG), which is a DNA adduct generated from acetaldehyde, was studied by the addition reaction of acetaldehyde-d(4 )to 2'-deoxyguanosine (dG) in deuterium oxide (D2O), with the aim to obtain an isotope internal standard for the liquid chromatography/tandem mass spectrometry (LC/MS/MS) quantitation of Et-dG. The replacement of the dG C-8 hydrogen atom by a deuteron atom took place at 50 degrees C in D2O and afforded a mixture of Et-dG-d(4) and Et-dG-d(5). Et-dG-d(4), which was stable in aqueous solutions, was prepared by incubating the mixture in H2O at 60 degrees C for 48 h. The calibration curve was obtained by multiple reaction monitoring (MRM) measurements using a hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometric (HILIC/ESI-MS/MS) system between the Et-dG concentration, ranging from 1.0 x 10(-10) to 4.0 x 10(-9 )M in the sample solutions, and the relative peak areas of Et-dG (m/z: 296.1 -> 180.1) to the value of Et-dG-d(4) (m/z: 300.2 -> 184.2), with an internal standard showing good linearity (R-2 = 0.995, n = 5).